CN102321591A

CN102321591A - Assembly mechanism of virus-like particle of hepatitis E virus and preparation method thereof

Info

Publication number: CN102321591A
Application number: CN201110234805A
Authority: CN
Inventors: 夏宁邵; 李少伟; 杨春燕; 鲜阳凌; 杜海莲; 张军
Original assignee: Xiamen University; Xiamen Innovax Biotech Co Ltd
Current assignee: Xiamen University; Xiamen Innovax Biotech Co Ltd
Priority date: 2011-08-17
Filing date: 2011-08-17
Publication date: 2012-01-18
Anticipated expiration: 2031-08-17
Also published as: CN102321591B

Abstract

The invention illustrates an assembly mechanism of a virus-like particle (VLP) of hepatitis E virus (HEV), and provides a method for assembling the VLP by HEV capsid protein or its variants or fragments. In addition, the invention also provides a method for controlling the assembly of the HEV VLP, which controls the assembly of HEV capsid protein or its variants or fragments to the VLP by controlling the salt concentration and/or temperature.

Description

Composition mechanism of the virus-like particle of hepatitis E virus and preparation method thereof

Technical field

The present invention relates to molecular biology and field of virology.Especially; The present invention has illustrated hepatitis E virus (Hepatitis E Virus; Virus-like particle HEV) (virus-like particle, composition mechanism VLP), and a kind of method that is used for HEV capsid protein or its variant or slice groups are dressed up VLP is provided.In addition, the present invention also provides the method for the assembling that is used to control HEVVLP, and it controls the assembling to VLP of HEV capsid protein or its variant or fragment through control salt concn and/or temperature.

Background technology

Hepatitis E is that (Hepatitis E Virus HEV) infects and to cause, and it is one of main viral hepatitis, and majority is self limiting by hepatitis E virus.The symptom and the hepatitis A of hepatitis E are similar, but case fatality rate is higher, and symptom is heavier: pregnant woman's case fatality rate can be up to 20%, and chronic hepatitis patients merges HEV and infect and be prone to cause liver failure, and mortality ratio reaches 70%.

HEV mainly passes through intestinal transmitted, but also can propagate through blood transfusion or vertical approach, usually cause large-scale eruption and prevalence.Since nearly half a century, document has been put down in writing nearly ten of the hepatitis E great outbursts more than ten thousand examples, and wherein biggest one betides the Chinese Xinjiang of 1986-1988,119,280 examples of falling ill altogether, and dead 707 people are comprising 414 pregnant woman.Because people are very limited to the research of HEV, therefore hepatitis E is in a kind of unheeded state for a long time.Got into since 21 century, along with the sensitivity and specific the increasing substantially of hepatitis E diagnostic reagent, increasing hepatitis E case is made a definite diagnosis.According to statistics, the population in the whole world nearly 1/3rd once infected HEV.

More and more evidences shows, hepatitis E is a kind of people beast disease that takes a disease altogether.Pig is the main animal host of HEV, and is the important contagium of human hepatitis E.Chinese scholar is to 9 of 120 pig farms of 20 provinces and cities, the whole nation and importquarantine; The HEV infection conditions of 055 pig has been carried out serosurvey; The result confirms that the HEV in the Chinese commodity pig infects extremely common; Total infection rate is up to 83%, and from domestic a plurality of geographic pigs, has been separated to the HEV virus strain.

Hepatitis E virus (HEV) is a kind of RNA viruses of sub-thread normal chain, and its capsid is the icosahedron symplex structure of the T=3 that dressed up by the single structure protein groups, the genome of the about 7.2kb of parcel.The diameter of HEV virion is about 27～34nm, no coating.The HEV genome comprises 5 ' end cap shape structure and poly A tract; And comprise three overlapped open reading frame (ORF:ORF1; ORF2 and ORF3), its both sides be respectively 5 of a weak point ' end non-coding region (5 ' UTR) with one by the short 3 ' end non-coding region (3 ' UTR) of polyadenylic acid terminated.ORF1 is about 5kb, is positioned at 5 ' end of coding region, is the maximum opening code-reading frame of HEV, and it mainly act as: in the presence of RNA synthetic enzyme, coding duplicates relevant Nonstructural Protein (pORF1) with viral RNA.ORF2 is about 2kb, is positioned at 3 ' end of coding region, is the primary structure gene coding region of HEV, and it mainly act as: coding viral capsid proteins pORF2---a kind ofly contain 660 amino acid whose glycosylated proteins.Recently also combine activity to ORF2 albumen with the special RNA of HEV genome 5 ' end and study, the result shows that both interactions have the formation that helps viral capsid.ORF 3 is little opening code-reading frames, and function is not illustrated as yet.The research prompting is arranged at present, and HEV ORF3 albumen possibly play an important role at aspects such as cell signalling, the assembling of HEV particle, release and immunity.

According to the homology of ORF2 structural protein gene, HEV can be divided into 4 main genotype at least.Molecule epidemic disease-ology research shows that HEV gene 1 type and 2 types mainly infect the mankind, often cause eruption and prevalence; Gene 3 types and 4 types are the infection animal and the mankind simultaneously, are sporadic popular.The HEV representative strains of four kinds of types is respectively Burma's strain, Mexico's strain, U.S.'s strain and Chinese variant.At present, chicken HEV is classified as gene 5 types.Various HEV has higher homology on the ORF2 aminoacid sequence, this causes on the HEV serology HEV all over the world to be same serotype.But the cross protection between each genotype virus has been simplified the difficulty of vaccine development greatly.

At present, the cell cultures of HEV and tissue culture all fail to succeed.Yet, since the virus-like particle of HEV (virus-like particle VLP) has good immunoreactivity and immunogenicity, can be used for developing the recombined hepatitis E hepatitis vaccine.Therefore, both at home and abroad the development of hepatitis E vaccine is mainly following: obtain structural protein through various expression systems, then in vivo or external the structural protein assembling is become virus-like particle (VLP), thereby prepare the VLP vaccine.At present; The report that is used for vaccine research about HEV VLP comprises as follows: (1) is through utilizing baculovirus/insect cell expression HEV ORF2 fragment aa112-aa 07; Can in cell lysate, obtain the HEV VLP (Robinson etc. of self-assembly in the body; Protein Expr Purif.1998,12 (1): 75-84); (2) utilize escherichia coli expression HEV ORF2 fragment aa368-aa606, it can be self-assembled into (Li etc., Vaccine.2005,23:2893-2901 into HEV VLP external; Li etc., JBC.2005,28 (5): 3400-3406).

Up to now; The hepatitis E vaccine that gets into clinical trial in the world has two kinds: a kind of is VLP vaccine by the HEV ORF2 fragment aa112-606 that comprises baculovirus/insect cell expression of GSK company development; It is compeled you the Buddhist nun and has got into the II clinical trial phase; Shown good immunogenicity, yet because a variety of causes, this research is interrupted; Another kind is the VLP vaccine that comprises the HEV ORF2 fragment aa368-aa606 of escherichia coli expression, and it has accomplished the III clinical trial phase, has shown good security and protectiveness (Zhu etc., Lancet, 2010).This shows that HEV VLP has good immunoreactivity and immunogenicity, can be used for developing the hepatitis E vaccine, is the ideal form of hepatitis E vaccine.

Therefore, the assembling of HEV VLP and regulatory mechanism have important theory and actual directive significance for the correlative study of hepatitis E virus and the production of hepatitis E vaccine.The present invention has illustrated the composition mechanism of HEV VLP through experiment, and a kind of method that is used for HEV capsid protein or its variant are assembled into VLP is provided on this basis, and the new method for preparing HEV VLP is provided.

Summary of the invention

The explanation of the relational language among the present invention and explanation

In the present invention, except as otherwise noted, otherwise the Science and Technology noun that uses among this paper has the implication of those skilled in the art institute common sense.And analytical chemistry, biological chemistry, cell cultures, Immunology Lab operation steps used among this paper are widely used conventional steps in the corresponding field.Simultaneously, in order to understand the present invention better, the definition and the explanation of relational language are provided below.

According to the present invention, term " escherichia expression system " is meant the expression system of being made up of intestinal bacteria (bacterial strain) and carrier, and wherein intestinal bacteria (bacterial strain) derive from available bacterial strain on the market; Such as but not limited to: GI698, ER2566, BL21 (DE3); B834 (DE3), BLR (DE3).

According to the present invention, term " carrier (vector) " is meant, can polynucleotide be inserted a kind of nucleic acid vehicle wherein.When carrier can make the albumen of the polynucleotide encoding of insertion obtain to express, carrier was called expression vector.Carrier can be through transforming, and transduction or transfection import host cell, makes its genetic material element that carries in host cell, obtain to express.Carrier is well known to a person skilled in the art, includes but not limited to: plasmid; Phage; Coemid or the like.

According to the present invention, term " HEV ORF2 " is meant the genomic ORF2 of hepatitis E virus (HEV), and its sequence is well known in the art (referring to, DDBJ data logging number for example: D11092), and the capsid protein of coding HEV.In the present invention, when relating to the sequence of HEV ORF2, it uses DDBJ data logging number: the sequence shown in the D11092 is described.For example; Statement " the 368-606 amino acids residue of HEV ORF2 encoded polypeptides " (also is abbreviated as in this article; HEV ORF2 fragment aa 368-aa 606 or HEV ORF2aa 368-aa 606) in 368-606 amino acids residue be meant the 368-606 amino acids residue of D11092 encoded polypeptides.Yet; It will be appreciated by those skilled in the art that in HEV ORF2 or its encoded polypeptides, can natural generation or artificially introduce sudden change or variation (includes but not limited to; Displacement; Disappearance and/or interpolation), and do not influence its biological function (promptly in the present invention, coding can form the capsid protein of HEV VLP).Therefore, in the present invention, term " HEV ORF2 " should comprise all these type of sequences, for example comprise the sequence shown in the D11092 with and natural or artificial variant.And when describing the sequence fragment of HEV ORF2 (or its encoded polypeptides), the sequence fragment that it not only comprises D11092 (or its encoded polypeptides) also comprises the corresponding sequence fragment in the natural or artificial variant of D11092 (or its encoded polypeptides).For example, statement " the 368-606 amino acids residue of HEVORF2 encoded polypeptides " comprises the 368-606 amino acids residue of D11092 encoded polypeptides, and the respective segments in the variant of D11092 encoded polypeptides (natural or artificial).According to the present invention, statement " corresponding sequence fragment " or " respective segments " is meant, when sequence being carried out optimum when comparison, promptly when sequence is compared with the highest percentage ratio identity of acquisition, is positioned at the fragment of equivalent site in the sequence that compares.

According to the present invention, term " HEV capsid protein " is meant that by HEV ORF2 encoded protein, it can be assembled into the HEV virus-like particle.

According to the present invention, when using in the background at albumen/polypeptide, term " variant " is meant such albumen, its aminoacid sequence with reference to albumen/polypeptide (for example; HEV capsid protein of the present invention) amino acid difference (for example, conservative amino acid replacement) that aminoacid sequence has one or more (for example 1-10 or 1-5 or 1-3) perhaps has at least 60%; 80%, 85%, 90%; 95%, 96%, 97%; 98%, or 99% identity, and it has kept the necessary characteristic with reference to albumen/polypeptide.In the present invention, the necessary characteristic of albumen/polypeptide (for example, HEV capsid protein of the present invention) can refer to have the ability that is assembled into the HEV virus-like particle.

According to the present invention, term " identity " be used in reference between two polypeptide or two nucleic acid between the match condition of sequence.When certain position in two sequences that compare is all occupied by identical base or amino acid monomer subunit (for example; Certain position in each of two dna moleculars is all occupied by VITAMIN B4; Or certain position in each of two polypeptide is all occupied by Methionin), each molecule is same on this position so." percentage ratio identity " between two sequences is by the function of the total matched position number of these two sequences divided by position number * 100 that compare.For example, if in 10 positions of two sequences 6 couplings are arranged, these two sequences have 60% identity so.For example, dna sequence dna CTGACT and CAGGTT have 50% identity (in 6 positions 3 location matches being arranged altogether).Usually, two sequence alignments are being compared when producing maximum identity.Such comparison can be through using, for example, can through computer program for example the Align program (DNAstar, the method for people such as Needleman (1970) J.Mol.Biol.48:443-453 that Inc.) carries out easily realizes.Also can use the E.Meyers and W.Miller (the Comput.Appl Biosci. of the ALIGN program that has been integrated into (version 2 .0); 4:11-17 (1988)) algorithm uses PAM120 weight residue table (weight residue table), 12 notch length point penalty and 4 breach point penalty to measure two percentage ratio identity between the aminoacid sequence.In addition; Can use Needleman and Wunsch (J MoI Biol.48:444-453 (1970)) algorithm in the GAP program that has been integrated into GCG software package (can on www.gcg.com, obtain), use Blossum 62 matrixes or PAM250 matrix and 16,14,12,10,8,6 or 4 breach weight (gap weight) and 1,2,3,4,5 or 6 length weight to measure two percentage ratio identity between the aminoacid sequence.

Like what use among this paper, term " conservative substitution " means the amino-acid substitution that can influence sharply or change the biological function of the albumen/polypeptide that comprises aminoacid sequence.For example, can for example site-directed mutagenesis and PCR mediated mutagenesis be introduced conservative substitution through standard technique known in the art.Conservative amino acid replacement comprises the displacement that substitutes amino-acid residue with the amino-acid residue with similar side chain; For example be used on the physics or displacement that the residue of similar with corresponding amino-acid residue on the function (for example have similar size, shape, electric charge, chemical property, comprise the ability that forms covalent linkage or hydrogen bond etc.) carries out.In this area, defined the family of amino-acid residue with similar side chain.These families comprise (for example having basic side chain; Methionin, l-arginine and Histidine), acid side-chain (for example aspartic acid, L-glutamic acid), uncharged polar side chain (for example glycocoll, l-asparagine, Stimulina, Serine, Threonine, tyrosine, halfcystine, tryptophane), non-polar sidechain (for example L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine(Phe), methionine(Met)), β branched building block (for example; Threonine, Xie Ansuan, Isoleucine) and the amino acid of aromatic series side chain (for example, tyrosine, phenylalanine(Phe), tryptophane, Histidine).Therefore, preferably use another amino-acid residue to substitute corresponding amino-acid residue from same side chain family.The method of identifying conservative aminoacid substitutions in this area be know (referring to, for example, people such as Brummell, Biochem.32:1180-1187 (1993); People Protein Eng.12 (10): 879-884 (1999) such as Kobayashi; With people Proc.Natl Acad.Set USA 94:412-417 (1997) such as Burks, it incorporates this paper by reference into).

According to the present invention; Term " p239 " is meant HEV ORF2 fragment aa368-aa606 (it also comprised the methionine(Met) by the initiator codon coding before aa368); It has the ability that assembling becomes HEV VLP; Can be used for preparing the hepatitis E vaccine, and can be used for studying the composition mechanism of HEV VLP.The exemplary amino acid sequence of p239 polypeptide is shown in SEQ ID NO:2, and its exemplary encoding sequence is shown in SEQ ID NO:1.

According to the present invention, term " E2 " is meant, HEV ORF2 fragment aa394-aa606 (it also comprised the methionine(Met) by the initiator codon coding before aa394), and it does not have the ability that assembling becomes HEV VLP.The exemplary amino acid sequence of E2 polypeptide is shown in SEQ ID NO:4, and its exemplary encoding sequence is shown in SEQ ID NO:3.

According to the present invention, term " virus-like particle (VLP) " be meant do not contain viral nucleic acid, the hollow shell structure similar structurally with natural virion, it is made up of viral capsid proteins or its variant or fragment usually.Because VLP is structurally quite similar with natural virion, therefore, it has very strong immunogenicity and immunoreactivity.Simultaneously, because VLP does not contain the genetic material of virus, therefore, it does not have infectivity.Because above-mentioned advantage, VLP by exploitation as vaccine, some of them VLP vaccine has been successfully applied to clinical, for example but be not limited to: the tetravalence VLP Gardasil vaccine of Merck, p239VLP vaccine or the like.VLP structurally allows the insertion of foreign gene or gene fragment and forms mosaic type VLP, and can exogenous antigen be illustrated in its surface.In addition, most of VLP also has parcel nucleic acid or other micromolecular abilities, and therefore, it also can be used as the vehicle of gene or medicine.Virus-like particle also abbreviates particle in this application as.Like what use among the present invention, when protein can assembling assembly virus-like particle under the condition that does not have the sex change factor, this protein was called as " ability with assembling assembly virus-like particle ".In the present invention, " sex change factor " is meant, can make the factor of protein generation sex change, and it includes but not limited to, high temperature (for example, heating), denaturing agent (for example urea) or the like.

Many viral capsid proteins or its variant or fragment all have the ability of assembling assembly virus-like particle.Yet being not all capsid protein or its variant or fragment can both assembling assembly virus-like particle, for example E2 albumen.Can identify capsid protein or its variant or the fragment of ability through methods known in the art with assembling assembly virus-like particle.For example; Can confirm whether capsid protein or its variant or fragment have the ability of assembling assembly virus-like particle through following step: at room temperature capsid protein or its variant or fragment are placed the solution that does not contain denaturing agent, detect the existence of virus-like particle then.

According to the present invention, term " hydrophobic interaction " is meant, a kind of weak, the non-covalent interaction between the non-polar molecule (for example, hydrophobic amino acid residue).These non-polar molecules have the water of avoiding and mutual accumulative tendency in aqueous environment.Hydrophobic interaction is that the repulsive interaction through hydrophobic molecule and water takes place.This effect makes hydrophobic molecule draw close each other, and water is concentrated mutually, thus structurizing to a greater degree.Hydrophobic interaction is very crucial to the structure and the character of most protein.For example, protein assembles most of hydrophobic amino acid residue through hydrophobic interaction each other.

According to the present invention; Term " assembling " be meant virus structural protein (for example capsid protein) between or between structural protein and the nucleic acid through various interactions; Form the process of well-regulated grainy texture, it comprises the assembling of assembling of natural viral particulate and virus-like particle.

According to the present invention; Term " protein denaturation (denaturation) " is meant; Protein is because of receiving the influence of some physics or chemical factor, and the space conformation of molecule is destroyed, thereby causes its physico-chemical property to change and lose the phenomenon of original BA.Denaturation does not cause the destruction of prlmary structure of protein, but causes the destruction of the higher structure that secondary structure is above, and the protein after the sex change is called metaprotein.

If the sex change condition is acutely lasting, cause proteinic space conformation and BA irreversibly to be destroyed, so proteinic sex change is irreversible (that is irreversible denaturation).If the sex change condition is inviolent, metaprotein can recover its native conformation and BA under certain condition, and so proteinic sex change is reversible (that is a reversible denaturation).Under the situation of reversible denaturation, the variation of protein molecule internal structure is little.At this moment, if remove the sex change factor, denatured protein will recover its native conformation and BA so, and this phenomenon is called protein renaturation (renaturation).The instance of protein reversible sex change and renaturation is exemplified below: when stomach en-is heated to 80-90 ℃; It loses solvability; The ability of also not having digesting protein, however if temperature is reduced to 37 ℃ again, it can recover the ability of solvability and digesting protein again.

According to the present invention, the whole bag of tricks that the fragmentation of host cell can be known by one of skill in the art realizes, includes but not limited to that homogenizer is broken, clarifixator is broken, ultrasonication, grinding, high-pressure extrusion, N,O-Diacetylmuramidase handle or the like.

According to the present invention, various damping fluids are well known in the art, include but not limited to phosphate buffered saline buffer or the like.

The salt that can be used in the method for the present invention includes but not limited to hydrogen salt, subsalt, neutral salt, for example an alkali metal salt, alkaline earth salt, ammonium salt, hydrochloride, vitriol, supercarbonate, phosphoric acid salt or hydrophosphate, particularly NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄In one or more.According to the present invention, preferred especially salt is (NH ₄) ₂SO ₄

According to the present invention, term " polypeptide " has identical implication with " protein ", interchangeable use.According to the present invention, amino acid is abridged with single-letter well known in the art and trigram usually and is represented.For example, L-Ala can be represented with A or Ala.

According to the present invention; Term " pharmaceutically acceptable carrier and/or vehicle " is meant carrier and/or vehicle compatible with activeconstituents with the experimenter on pharmacology and/or physiology; It is well known in the art (referring to for example Remington ' s Pharmaceutical Sciences.Edited by Gennaro AR, 19th ed.Pennsylvania:Mack Publishing Company, 1995); And include but not limited to: the pH regulator agent; Tensio-active agent, adjuvant, ionic strength toughener.For example, the pH regulator agent includes but not limited to phosphate buffered saline buffer; Tensio-active agent comprises but is not limited to positively charged ion, negatively charged ion or non-ionics, for example Tween-80; Adjuvant includes but not limited to aluminium adjuvant (for example white lake), freund's adjuvant (for example complete Freund's adjuvant); The ionic strength toughener includes but not limited to sodium-chlor.

Those skilled in the art know, and HEV capsid protein (comprising its variant or fragment) can not be assembled into HEV VLP under the situation that denaturing agent (for example urea) exists.Under common situation, need denaturing agent be removed, the HEV capsid protein can be assembled into VLP.Yet; The contriver unexpectedly finds: HEV capsid protein or its variant or fragment are (for example; P239 albumen) in solution, exist under the situation of denaturing agent (for example, 4M urea), when in solution, adding certain density salt (for example 0.3M ammonium sulfate) at least; Can be self-assembled into VLP, and need not the denaturing agent in the solution is removed.In addition, the contriver finds that also the adding of ammonium sulfate helps HEV capsid protein or its variant or fragment to form the VLP of homogeneous more, and the assembling speed of VLP receives salt concn and Influence of Temperature in the solution.

Therefore; In one aspect; The invention provides the method for the virus-like particle (VLP) of preparation hepatitis E virus (HEV); It comprises step: under the condition that denaturing agent exists, make HEV capsid protein or its variant or slice groups dress up VLP through adding salt, wherein said HEV capsid protein or its variant or fragment have the ability that is assembled into VLP.

In a preferred embodiment, said denaturing agent for example can be urea, urea, Guanidinium hydrochloride or its combination.In a further preferred embodiment, said denaturing agent is a urea, for example 4M urea.

In a preferred embodiment, said salt for example can be NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, and particularly preferably be (NH ₄) ₂SO ₄In a further preferred embodiment, the ionic strength of said salt is 0.9mo l/kg at least.For example, said salt can be (the NH of 0.3M at least ₄) ₂SO ₄

In a preferred embodiment, said HEV capsid protein or its variant or fragment for example can be with HEV capsid protein or its variant or the fragment of inclusion body formal representation in intestinal bacteria.

In another preferred embodiment, the variant of said HEV capsid protein or fragment for example can be p239 albumen.Preferably, the proteic aminoacid sequence of said p239 is shown in SEQ IDNO:2.

In yet another aspect, the invention provides the method for the virus-like particle (VLP) of preparation hepatitis E virus (HEV), it comprises the steps:

1) HEV capsid protein or its variant or the fragment with the ability that is assembled into VLP is provided;

2) the HEV capsid protein of step 1) or its variant or fragment are dissolved in the solution that contains denaturing agent;

3) to step 2) add salt in the solution that obtains, so that said HEV capsid protein or its variant or slice groups are dressed up VLP;

Randomly, before carrying out step 3), the HEV capsid protein in containing the solution of denaturing agent or its variant or fragment are carried out purifying, for example, carry out purifying through anion-exchange chromatography and/or hydrophobic interaction chromatography;

Randomly, after step 3), remove the denaturing agent in the solution, for example carry out dialysis in the PBS solution and remove denaturing agent through the solution that will contain HEV VLP.

In a preferred embodiment, in step 1), for example through with the inclusion body formal representation said HEV capsid protein or its variant or fragment being provided in intestinal bacteria with it.

In a preferred embodiment, the variant of said HEV capsid protein or fragment are p239 albumen.Preferably, the proteic aminoacid sequence of said p239 is shown in SEQ ID NO:2.

In a preferred embodiment, in step 2) in, said denaturing agent for example can be urea, urea, Guanidinium hydrochloride or its combination.In a further preferred embodiment, said denaturing agent is a urea, for example 4M urea.

In a preferred embodiment, in step 3), said salt for example can be NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, and particularly preferably be (NH ₄) ₂SO ₄In a further preferred embodiment, the ionic strength of said salt is 0.9mol/kg at least.For example, said salt can be (the NH of 0.3M at least ₄) ₂SO ₄

As well known to those skilled in the art, the HEV virus-like particle possesses good immunoreactivity and immunogenicity, can be used for developing the recombined hepatitis E hepatitis vaccine.Therefore, in one aspect, the present invention also provides preparation to be used to prevent and/or treat the method for the vaccine of hepatitis E, and it comprises:

1) prepares the HEV virus-like particle according to the method for the invention;

2) with the HEV virus-like particle that is obtained and pharmaceutically acceptable carrier and/or excipient composition, thus the preparation vaccine.

In yet another aspect, the present invention also provides the vaccine that is used to prevent and/or treat hepatitis E, and it obtains according to aforesaid method of the present invention, or comprises the HEV virus-like particle for preparing according to the method for the invention and get.Randomly, vaccine of the present invention can also comprise pharmaceutically acceptable carrier and/or vehicle.

In yet another aspect; The invention provides control HEV capsid protein or its variant or slice groups and dress up the method for the assembling speed of VLP; It comprises step: control comprises the salt concn of HEV capsid protein or its variant or segmental solution and/or controls the temperature of said solution, and wherein said HEV capsid protein or its variant or fragment have the ability that is assembled into VLP.

In a preferred embodiment, said solution comprises or does not comprise denaturing agent.In a further preferred embodiment, said denaturing agent for example can be urea, urea, Guanidinium hydrochloride or its combination.In a further preferred embodiment, said denaturing agent is a urea, for example 4M urea.

In a preferred embodiment, the salt that comprises of said solution is NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, preferred (NH ₄) ₂SO ₄

In a preferred embodiment, when the salt concn of said solution raises and/or the temperature of said solution when raising, said assembling speed is accelerated.

In yet another aspect, the invention provides salt and be used to promote to have HEV capsid protein or its variant of the ability that is assembled into VLP or the purposes that slice groups is dressed up VLP.

In a preferred embodiment, said salt for example can be NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, and particularly preferably be (NH ₄) ₂SO ₄In a further preferred embodiment, the ionic strength of said salt is 0.9mol/kg at least.For example, said salt can be (the NH of 0.3M at least ₄) ₂SO ₄

In a preferred embodiment, said salt is used to promote the assembling of VLP under denaturing agent existence or non-existent condition.In a further preferred embodiment, said denaturing agent for example can be urea, urea, Guanidinium hydrochloride or its combination.In a further preferred embodiment, said denaturing agent is a urea, for example 4M urea.

In yet another aspect; The invention provides the method for destroying HEV capsid protein or its variant or segmental particle assembling ability, it comprises the hydrophobic amino acid that is arranged in the zone corresponding with HEV ORF2 aa368-395 in HEV capsid protein or its variant or the fragment is sported hydrophilic amino acid.

In a preferred embodiment, the variant of said HEV capsid protein or fragment comprise the corresponding zone with HEV ORF2 aa368-395.In a further preferred embodiment, the variant of said HEV capsid protein or fragment are p239 albumen.Preferably, the proteic aminoacid sequence of said p239 is shown in SEQ ID NO:2.

In a preferred embodiment, the said hydrophobic amino acid that is arranged in the zone corresponding the 372nd, 375 or 395 leucine that can be HEV ORF2 with HEV ORF2 aa368-395.In a further preferred embodiment, said leucine is sported L-glutamic acid.

In yet another aspect; The invention provides the method that promotes HEV capsid protein or its variant or slice groups to dress up VLP, it comprises the hydrophilic amino acid that is arranged in the zone corresponding with HEV ORF2 aa368-395 in HEV capsid protein or its variant or the fragment is sported hydrophobic amino acid.

As employed among the present invention; Statement " with the corresponding zone of HEV ORF2aa 368-395 " is meant; When sequence and HEV ORF2 encoded polypeptides are carried out the optimum comparison; Promptly when comparing when obtaining the highest percentage ratio identity, be in the zone of equivalent site in the sequence that compares with HEV ORF2aa368-395.

The beneficial effect of the invention

The expression system that is used to prepare the HEV virus-like particle at present can be divided into eukaryotic expression system and prokaryotic expression system.

The HEV capsid protein native conformation of in eukaryotic expression system, expressing destroys few, and the spontaneous formation VLP of ability is the VLP with correct conformation after often expressing.But at present eukaryotic expression system for example baculovirus expression system exist expression amount low with yeast expression system, cultivate defectives such as cost height, brought very big difficulty to large-scale industrial production, and be unfavorable in cell, studying the composition mechanism of VLP.

In prokaryotic expression system, escherichia expression system has that to cultivate cost low, the expression amount advantages of higher.Yet, often lose correct native conformation at the albumen of expression in escherichia coli, with the inclusion body formal representation in the deposition in.At present to carry out that renaturation assembles with particle be a difficult problem to being expressed in albumen in the inclusion body.The VLP that renaturation difficulty and inefficiency make from inclusion body acquisition have correct conformation is difficult in scale operation, implement, and can only be confined in the small-scale laboratory study.

The present invention provides the new method that the HEV capsid protein is assembled into VLP on the basis of illustrating the mechanism that the HEV capsid protein is assembled into VLP, solved the problems referred to above effectively.At first, the present invention uses escherichia expression system to express HEV capsid protein (p239 albumen), has guaranteed higher expression amount.Secondly; Method of the present invention makes it possible to make simply, quickly and easily the HEV capsid protein that is expressed in the inclusion body to carry out renaturation and particle assembling; And the HEV VLP that obtains of assembling is verified to have good immunoreactivity and immunogenicity; Similar with the natural viral particle form, be the good vaccine form that is suitable for scale operation.The 3rd, provided by the present invention that the HEV capsid protein is assembled into the method for VLP is simple to operate, and good reproducibility can be applicable to large-scale industrial production.Therefore, method of the present invention makes the large-scale industrial production that escherichia expression system is applied to the hepatitis E vaccine become possibility.

In addition, the present invention confirms, through control salt concn and/or temperature, can control the assembling to VLP of HEV capsid protein or its variant, and confirms, under the situation that certain density salt (for example, 0.3M ammonium sulfate) exists, the HEV VLP that assembling obtains is homogeneous more.This control and stdn for the large-scale industrial production of hepatitis E vaccine is very favourable.

At last, HEV VLP of the present invention can assemble under the situation that denaturing agent (for example, urea) exists and obtain.Therefore, new VLP assemble method provided by the present invention for control VLP assembling provides a kind of new thinking, also provides a kind of brand-new visual field for proteinic gathering.

To combine accompanying drawing and embodiment that embodiment of the present invention are described in detail below, but it will be understood by those skilled in the art that attached drawings and embodiment only are used to explain the present invention, rather than to the qualification of scope of the present invention.According to the following detailed description of accompanying drawing and preferred embodiment, it is obvious that various purposes of the present invention and favourable aspect will become to those skilled in the art.

Description of drawings

Fig. 1: derived from the p239 and the form of E2 (reference protein) in 4M urea of hepatitis E virus capsid protein.Figure 1A has shown the subsidence rate result of experiment of p239 in 4M urea; Figure 1B has shown the subsidence rate result of experiment of E2 in 4M urea; Fig. 1 C has shown the sedimentation equilibrium result of experiment of p239 in 4M urea; Fig. 1 D has shown the sedimentation equilibrium result of experiment of E2 in 4M urea.Can know that from Fig. 1 under the situation that does not have salt, p239 and E2 all present one-component in 4M urea, are the dimeric forms of homogeneous, can not be assembled into VLP.

Fig. 2: the mensuration of hatching the settling ratio of p239 and E2 after 24 hours with the ammonium sulfate of different concns.P239 (Fig. 2 A-D) and E2 (Fig. 2 E-H) were hatched 24 hours in the 4M of the ammonium sulfate that contains different concns urea respectively, dialyse then to the PBS solution to carry out renaturation.Wherein, p239: Fig. 2 A, 0M (NH ₄) ₂SO ₄Fig. 2 B, 0.3M (NH ₄) ₂SO ₄Fig. 2 C, 0.5M (NH ₄) ₂SO ₄Fig. 2 D, 1.0M (NH ₄) ₂SO ₄E2: Fig. 2 E, 0M (NH ₄) ₂SO ₄Fig. 2 F, 0.3M (NH ₄) ₂SO ₄Fig. 2 G, 0.5M (NH ₄) ₂SO ₄Fig. 2 H, 1.0M (NH ₄) ₂SO ₄Can know that from Fig. 2 hatching of ammonium sulfate helps p239 formation VLP particle (Fig. 2 A-D), and to not influence (Fig. 2 E-H) of E2.In addition, present the components (Fig. 2 A) of two kinds of different sizes after the p239 renaturation without the ammonium sulfate processing, this shows that the processing of ammonium sulfate can impel p239 to form the more particle of homogeneous.

Fig. 3: hatch the state analysis of p239 in urea after 6 hours with the ammonium sulfate of different concns.Fig. 3 A, extreme ultraviolet circular dichroism spectral results, black line is unassembled p239, red line is at 4M urea+0.3M (NH ₄) ₂SO ₄The middle p239 that handles 6 hours, blue line are the p239 particle through assembling; Fig. 3 B, near ultraviolet circular dichroism spectral results, black line is unassembled p239, red line is at 4M urea+0.3M (NH ₄) ₂SO ₄The middle p239 that handles 6 hours, blue line are the p239 particle through assembling; Fig. 3 C, the sieve chromatography analytical results, 1 dark green line is unassembled p239,2 bright green lines are at 4M urea+0.3M (NH ₄) ₂SO ₄The middle p239 that handles 6 hours, 3 brown lines are at 4M urea+0.5M (NH ₄) ₂SO ₄The middle p239 that handles 6 hours, 4 pink lines are at 4M urea+1.0M (NH ₄) ₂SO ₄The middle p239 that handles 6 hours, 5 blue lines are the p239 particle through assembling; Fig. 3 D, the dynamic light scattering analytical results is at 4M urea+0.3M (NH ₄) ₂SO ₄The middle p239 that handles 6 hours.Can know that from Fig. 3 after in urea, adding certain density ammonium sulfate and hatching 6 hours, p239 promptly can be assembled into VLP, and need not to remove denaturing agent urea.This result shows, exists under the situation of denaturing agent, and the assembling of p239 can be induced through certain salt concn.

Fig. 4: different salt ions and of the influence of different salt concn to the proteic particle assembling of p239.Unassembled p239 albumen is joined the (NH that contains different concns (0.3M, 0.5M or 1.0M) ₄) ₂SO ₄, Na ₂SO ₄, NH ₄In the 4M urea of Cl or NaCl and hatched 6 hours, measure the settling ratio of p239 then.The result shows that the rising of ionic strength can promote the assembling of p239, and the assembling of p239 needs certain ionic strength, that is: at least about the ionic strength (ionic strength of 0.3M ammonium sulfate is 0.9mo l/kg) of 0.9mol/kg.

Fig. 5: p239 albumen is at (the NH that contains different concns (0.3M, 0.5M or 1.0M) ₄) ₂SO ₄4M urea in the dynamic process (analyzing hatching the 1st, 2,3,4,5,6,7 and 8 hour the time point in back) of assembling.Fig. 5 A is with 0.3M (NH ₄) ₂SO ₄, the assembling process of the p239 of 25 ℃ of processing; Fig. 5 B is with 0.3M (NH ₄) ₂SO ₄, the assembling process of the p239 of 37 ℃ of processing; Fig. 5 C is with 0.5M (NH ₄) ₂SO ₄, the assembling process of the p239 of 25 ℃ of processing; Fig. 5 D is with 0.5M (NH ₄) ₂SO ₄, the assembling process of the p239 of 37 ℃ of processing; Fig. 5 E is with 1.0M (NH ₄) ₂SO ₄, the assembling process of the p239 of 25 ℃ of processing; Fig. 5 F is with 1.0M (NH ₄) ₂SO ₄, the assembling process of the p239 of 37 ℃ of processing; Fig. 5 G, control sample E2 albumen is containing 0.0M, 0.3M, 0.5M or 1.0M (NH ₄) ₂SO ₄4M urea in 37 ℃ of subsidence rate analyses after hatching 24 hours.As can be seen from Figure 5, the assembling speed of p239 and temperature and (NH ₄) ₂SO ₄Concentration is proportionate: concentration is high more, and assembling speed is fast more, and temperature is high more, and assembling speed is also fast more.

Fig. 6: p239 in the urea of the ammonium sulfate that contains different concns, the proximate analysis of the assembling dynamic process under differing temps.Fig. 6 A is at 25 ℃ of incubation temperature and different (NH ₄) ₂SO ₄Under the concentration, the component in the p239 assembling process distributes; Fig. 6 B is at 37 ℃ of incubation temperature and different (NH ₄) ₂SO ₄Under the concentration, the component in the p239 assembling process distributes; Fig. 6 C is at 25 ℃ of incubation temperature and different (NH ₄) ₂SO ₄Under the concentration, the change curve of each component in the p239 assembling process; Fig. 6 D is at 37 ℃ of incubation temperature and different (NH ₄) ₂SO ₄After hatching 24hr under the concentration, the distribution range of p239 particle and the proteic settling ratio of E2.Can find out from Fig. 6 A-6C, along with the increase or the (NH of temperature ₄) ₂SO ₄The raising of concentration, the assembling speed of p239 is accelerated.Can find out (NH from Fig. 6 D ₄) ₂SO ₄Concentration is consistent to the trend that influences of p239 particle and the proteic settling ratio of E2: along with (NH ₄) ₂SO ₄The raising of concentration, settling ratio descends.

Fig. 7: the analysis of the final assembled state of in 4M urea, assembling of p239 particulate.P239 albumen is containing 0.5M (NH ₄) ₂SO ₄Urea under 37 ℃, hatched 12 hours, to be assembled into the p239 particle, be dialyzed to then in the PBS solution, thereby obtain the final assembled state of p239 particulate.Fig. 7 A, the TEM analysis of the final assembled state of p239 particle, Bar=100nm; Fig. 7 B, the subsidence rate analysis of the final assembled state of p239 particle; Fig. 7 C, the molecular sieve high-efficient liquid-phase chromatographic analysis of the final assembled state of p239 particle; Fig. 7 D, the dynamic light scattering analysis of the final assembled state of p239 particle.As can beappreciated from fig. 7, the p239 protein groups has been dressed up the HEV virus-like particle of homogeneous.

Fig. 8: when the p239 protein groups is dressed up to virus-like particle in the interaction of three times of shaft positions.Fig. 8 A, the interaction of the alpha-helix of three times of shaft positions; Fig. 8 B, the partial enlarged drawing of three times of shaft positions, it shows that the 397th of HEV capsid protein and the 443rd tyrosine residues side chain have significant contribution in interaction.

Fig. 9: the N end structure domain model of the trimerizing intermediate state that p239 albumen forms in assembling process.Fig. 9 A, the skeleton of p239 albumen n end structural domain; Fig. 9 B, the hydrophobic core that p239 albumen n end structural domain forms (blue surface), wherein the 372nd, 375 and 395 of the HEV capsid protein the leucine residue has significant contribution to hydrophobic interaction; Fig. 9 C, the structural models (seeing view inwardly) of p239 albumen trimerizing intermediate state; Fig. 9 D, the structural models (view looks out) of p239 albumen trimerizing intermediate state.Annotate: this model select from part corresponding in the HEV 3 C-type virus C appearance crystalline structure with p239 (said crystalline structure is numbered 2ZTN in the PDB DB, referring to Yamashita etc., Proc Natl Acad Sci USA, 2009,106:12986-12991).

Figure 10: the mutation analysis of p239 albumen n end structural domain.Figure 10 A, the SDS-PAGE and the Western engram analysis of p239 and 3 point mutation albumen (L372E, L375E and L395E) thereof; Figure 10 B, the sieve chromatography analysis of p239 and 3 point mutation albumen (L372E, L375E and L395E) thereof.As can beappreciated from fig. 10,3 point mutation albumen (L372E, L375E and L395E) all can not be assembled and become particle, and have lost the immunoreactivity with viral hepatitis type E reconvalescent serum.In Figure 10 A, N representes this sample for without the sample of heat treated, H represent this sample for incubation in boiling water bath 3 minutes sample; (+) representes that this sample comprises dimer or has monoclonal antibody reactive behavior and (-) and representes that this sample does not comprise dimer or do not have the monoclonal antibody reactive behavior.

Sequence information

The information of the sequence that the present invention relates to is provided in the following table 1.

Table 1: the description of sequence

Sequence 1 (SEQ ID NO:1):

ATGATAGCGCTTACCCTGTTTAACCTTGCTGACACCCTGCTAGGCGGTCTACCCACAGAATTGATTTCGTCGGCAGG

TGGACAGCTGTTCTACTCTCGTCCCGTTGTCTCGGCCAATGGCGAGCCGACTGTTAAGCTTTATACATCTGTAGAGA

ATGCTCAGCAGGATAAGGGTATTGCAATCCCGCATGACATCGACCTCGGGGAGTCTCGTGTAGTTATTCAGGATTAT

GACAACCAACATGAGCAGGACCGACCGACACCTTCCCCAGCCCCATCGCGCCCTTTTTCTGTCCTCCGAGCTAATGA

TGTGCTTTGGCTTTCTCTCACCGCTGCCGAGTATGACCAGTCCACTTACGGCTCTTCGACCGGCCCAGTCTATGTCT

CTGACTCTGTGACCTTGGTTAATGTTGCGACCGGCGCGCAGGCCGTTGCCCGGTCACTCGACTGGACCAAGGTCACA

CTTGATGGTCGCCCCCTTTCCACCATCCAGCAGCATTCAAAGACCTTCTTTGTCCTGCCGCTCCGCGGTAAGCTCTC

CTTTTGGGAGGCAGGTACTACTAAAGCCGGGTACCCTTATAATTATAACACCACTGCTAGTGACCAACTGCTCGTTG

AGAATGCCGCTGGGCATCGGGTTGCTATTTCCACTTACACCACTAGCCTGGGTGCTGGCCCCGTCTCTATTTCCGCG

GTTGCTGTTTTAGCCCCCCCTCCGCGCTAG

Sequence 2 (SEQ ID NO:2):

MIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDY

DNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVT

LDGRPLSTTQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISA

VAVLAPPPR

Sequence 3 (SEQ ID NO:3):

ATGCAGCTGTTCTACTCTCGTCCCGTTGTCTCGGCCAATGGCGAGCCGACTGTTAAGCTTTATACATCTGTAGAGAA

TGCTCAGCAGGATAAGGGTATTGCAATCCCGCATGACATCGACCTCGGGGAGTCTCGTGTAGTTATTCAGGATTATG

ACAACCAACATGAGCAGGACCGACCGACACCTTCCCCAGCCCCATCGCGCCCTTTTTCTGTCCTCCGAGCTAATGAT

GTGCTTTGGCTTTCTCTCACCGCTGCCGAGTATGACCAGTCCACTTACGGCTCTTCGACCGGCCCAGTCTATGTCTC

TGACTCTGTGACCTTGGTTAATGTTGCGACCGGCGCGCAGGCCGTTGCCCGGTCACTCGACTGGACCAAGGTCACAC

TTGATGGTCGCCCCCTTTCCACCATCCAGCAGCATTCAAAGACCTTCTTTGTCCTGCCGCTCCGCGGTAAGCTCTCC

TTTTGGGAGGCAGGTACTACTAAAGCCGGGTACCCTTATAATTATAACACCACTGCTAGTGACCAACTGCTCGTTGA

GAATGCCGCTGGGCATCGGGTTGCTATTTCCACTTACACCACTAGCCTGGGTGCTGGCCCCGTCTCTATTTCCGCGG

TTGCTGTTTTAGCCCCCCCTCCGCGCTAG

Sequence 4 (SEQ ID NO:4):

MQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRAND

VLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTTQQYSKTFFVLPLRGKLS

FWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPPPR

Embodiment

Existing the present invention is described with reference to the following embodiment that is intended to illustrate the present invention (and non-limiting the present invention).

Only if specialize, employed experimental methods of molecular biology and immunodetection among the present invention, basically with reference to people such as J.Sambrook, molecular cloning: laboratory manual; The 2nd edition, press of cold spring harbor laboratory, 1989; And people such as F.M.Ausubel, fine works molecular biology experiment guide, the 3rd edition; John Wiley&Sons, Inc., the method described in 1995 is carried out.Unreceipted actual conditions person among the embodiment all carries out according to the condition of the common known normal condition in this area or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.Those skilled in the art know, and embodiment describes the present invention with way of example, and are not intended to limit the present invention's scope required for protection.

Embodiment 1: derived from the state of p239 in urea of HEV capsid protein

According to method (Chinese invention patent: the ZL 02822218.0 that describes before; People such as Li, JBC, 2005; With people such as Li, Vaccine, 2005), clone, expression and purifying are derived from the p239 (HEV ORF2 aa368-606, its nucleotide sequence and aminoacid sequence are respectively shown in SEQ ID NO:1 and SEQ ID NO:2) of hepatitis E virus capsid protein.P239 albumen has kept the ability that is assembled into virus-like particle of HEV capsid protein, is used to analyze the composition mechanism of HEV VLP.P239 albumen mainly with the form of inclusion body at expression in escherichia coli.Behind the washing purifying; Most of p239 protein dissolution in the inclusion body is in the 4M urea soln; Use then anion-exchange chromatography and hydrophobic interaction chromatography in the solution that contains 4M urea with the purity more than the p239 protein purification to 95%, to be used for follow-up analysis.In addition, express with same method and purifying derived from the E2 albumen (HEV ORF2aa394-606, its nucleotide sequence and aminoacid sequence are respectively shown in SEQ ID NO:3 and SEQ IDNO:4) of HEV capsid protein.The E2 proteins lose be assembled into the ability (referring to people such as Li, JBC, 2005) of virus-like particle, with comparing.

According to the method for describing before, operational analysis type ultracentrifuge (XL-A type, U.S. Beckman company produces); Through sedimentation rate method and sedimentation equilibrium method (Schuck P etc.; Biophys J, 2000,78:1606-1619) measure the state of p239 in the 4M urea soln.Mensuration result is as shown in Figure 1.The result shows that p239 albumen presents one-component in the 4M urea soln, and its settling ratio is 1.7s, and frictional coefficient is 1.94 (Figure 1A), and molecular weight is 45772 ± 730Da (theoretical monomer molecule amount is 25521Da) (Fig. 1 C).This shows that p239 albumen is the single dimeric forms of component in the 4M urea soln, and the non-globular preteins of comparatively unfolding for structure (frictional coefficient of globular preteins is 1.2).Reference protein E2 also is the dimeric forms of homogeneous in the 4M urea soln, and its settling ratio is 2.0s, and frictional coefficient is 1.94 (Figure 1B), and molecular weight is 40981 ± 1523Da (theoretical monomer molecule amount is 23097Da) (Fig. 1 D).The The above results explanation, in the 4M urea soln, the proteic state of p239 is identical with E2 albumen, and the two is the dimeric forms of homogeneous, and does not have grain fraction.

The discovery of the key factor of embodiment 2:p239 particle assembling

When utilizing hydrophobic interaction chromatography that the p239 albumen in the 4M urea soln is carried out purifying, need to use ammonium sulfate to increase proteic hydrophobicity.Usually, when carrying out Phenyl FFSepharose chromatography, contain 1M ammonium sulfate in the sample solution, and the final solution that contains 0.1-0.2M ammonium sulfate of using carries out the proteic wash-out of p239.Behind wash-out, generally make the p239 protein renaturation and be assembled into particle through the denaturing agent urea of removing in the solution.In this application, the contriver is surprised to find that, the ammonium sulfate in the urea soln is increased to 0.3M at least; And after placing for some time; P239 albumen can be assembled into particle under the situation that urea exists, and need not to remove urea (that is, whether existing irrelevant with urea).It below is result of study to proteic composition mechanism of p239 and process.

The proteic renaturation of p239 receives the influence of the ammonium sulfate of different concns

Purified p239 albumen (in the 4M urea soln) is diluted to 1.0mg/ml, and in solution, adds (NH ₄) ₂SO ₄Until final concentration is 0M, 0.3M, 0.5M or 1M, and the concentration of urea keeps 4M constant.Place 37 ℃ to hatch respectively 24 hours each protein sample, then respectively to the PBS solution renaturation of dialysing.In addition, also handle E2 albumen in the same way, with comparing.After the processing, described in embodiment 1, measure each p239 sample and the state of E2 sample in PBS solution through sedimentation rate method.

The result is as shown in Figure 2.The result shows that the processing of ammonium sulfate has promoted the p239 particulate to form (Fig. 2 A-D).Especially, the p239 sample (that is 0M (NH, that handles without ammonium sulfate ₄) ₂SO ₄) present the components (Fig. 2 A) of two kinds of different sizes after the renaturation, show that the processing of ammonium sulfate can impel p239 to form the more particle of homogeneous; Through 0.5M (NH ₄) ₂SO ₄The p239 sample of handling presents the particle (Fig. 2 C) of homogeneous.By contrast, the E2 albumen of handling through the same manner all is rendered as single 2.7S component, and this shows that E2 albumen still is dimeric forms, and particle assembling (Fig. 2 E-H) does not take place.

D239 albumen is assembled into the particulate evidence in urea

As before described, utilize extreme ultraviolet circular dichroism (Circular Dichroism, CD) spectrum, near ultraviolet circular dichroism spectrum (Beychok S; 1966, Science, 154 (754): 1288-1299); Sieve chromatography is analyzed (Regnier FE, 1983, Science; 222 (4621): 245-252) with dynamic light scattering analysis (Bnachowicz E; 2006, Biochim Biophys Acta, 1764 (3): 405-413) etc. method detect p239 albumen in the 4M urea of the ammonium sulfate that contains different concns (0.3M, 0.5M or 1.0M) at 37 ℃ of states after hatching 6 hours.

At room temperature in CD spectrograph (J-810 type; Japan JASCO company produces) go up the p239 albumen (containing in the 4M urea of 0.3M ammonium sulfate) measuring unassembled p239 albumen (in 4M urea), handle through 0.3M ammonium sulfate and through the extreme ultraviolet and the near ultraviolet CD spectrum of the p239 of the tangential flow renaturation particle p239 particle of assembling (that is, through).Employed parameter is following: sensitivity 2m °/cm; Waveband width 1nm; Time constant 1sec; Sweep velocity 50nm/min.The CD data are with ellipse value (θ) expression, and unit is milli degree (mdeg).The employed sample concentration of extreme ultraviolet CD spectroscopic analysis is 4mg/ml, and scanning wavelength is 190-260nm, and the cuvette optical path is 0.01cm; The employed sample concentration of near ultraviolet CD spectrum is 0.5mg/ml, and scanning wavelength is 260-320nm, and the cuvette optical path is 1cm.

The result is as shown in Figure 3.The result of CD spectroscopic analysis shows; The proteic CD spectrum of p239 through ammonium sulfate is handled is more similar with the p239 particulate CD spectrum of warp assembling; And having significantly different (referring to Fig. 3 A and 3B) with the proteic CD spectrum of unassembled p239, the p239 albumen that this explanation is handled through ammonium sulfate is structurally more similar with the p239 particle.The result that sieve chromatography is analyzed shows; Through p239 sample that ammonium sulfate (0.3M, 0.5M or 1.0M) is handled under the situation that 4M urea exists; Formed the identical grain fraction of p239 particle of assembling with warp, it significantly is different from unassembled p239 albumen (referring to Fig. 3 C).The result that dynamic light scattering is analyzed shows, under the situation that 4M urea exists, formed the particle (referring to Fig. 3 D) of radius for about 20nm through p239 albumen that ammonium sulfate is handled.These evidences all show; In urea, add certain density ammonium sulfate and 37 ℃ hatch 6 hours after; P239 albumen promptly can be assembled into particle, and need not to remove denaturing agent urea (whether irrelevant with the existence of urea), and the proteic particle assembling of this explanation p239 can be induced through certain salt concn.

Different salt ions and of the influence of different salt concn to the proteic particle assembling of p239

In this experiment, further investigate various salt ((NH ₄) ₂SO ₄, Na ₂SO ₄, NH ₄Cl and NaCl) and salt concn (0.3M, 0.5M or 1.0M) to the influence of the proteic particle of p239 assembling.Particularly, unassembled p239 albumen is joined the (NH that contains different concns ₄) ₂SO ₄, Na ₂SO ₄, NH ₄In the 4M urea soln of Cl or NaCl, and hatched 6 hours in 37 ℃, described in embodiment 1, carry out the mensuration of settling ratio then, observe the influence that different salt ion and salt concn are assembled the proteic particle of p239.

The result is as shown in Figure 4.The result shows that different types of salt ion all can impel 239 albumen in urea, to be assembled into particle, could finally be assembled into particle but need reach certain ionic strength p239 albumen: 0.3M NaCl or NH ₄Cl can not lure that p239 albumen is assembled into and form particle that the p239 albumen in the solution is mainly the dimeric forms that settling ratio is 1.6S in urea; As NaCl or NH ₄When the concentration of Cl was increased to 1M, p239 albumen began assembling; 0.3M Na ₂SO ₄Or (NH ₄) ₂SO ₄Be enough to lure into that the assembling of p239 albumen forms particle; The The above results explanation, the proteic particle assembling of p239 needs certain ionic strength, that is: can impel the p239 protein groups to dress up particle more than or equal to the ionic strength of 0.9mol/kg.In addition, the result of Fig. 4 shows that also different anions there are differences the influence of particle assembling: SO ₄ ^2-Effect obviously be superior to Cl ^-

The dynamic process that p239 assembles in urea

With (NH ₄) ₂SO ₄Be added in p239 albumen/4M urea, make (NH ₄) ₂SO ₄Final concentration be respectively 0.3M, 0.5M and 1M; Final concentration of protein is 1.0mg/ml; It is constant that urea concentration is still kept 4M; Then it is placed respectively under 25 ℃ and 37 ℃ of two kinds of temperature and hatch, sampling in per 1 hour once obtains 8 samples (hatching the 1st, 2,3,4,5,6,7 and 8 hour the sample in back) altogether.According to the method for describing among the embodiment 1, all samples that obtains is carried out settling ratio measure, to analyze the dynamic process of the proteic particle assembling of p239.

The result is as shown in Figure 5.The result shows, (the NH of different concns ₄) ₂SO ₄Can lure that all p239 albumen assembles existing under the sex change condition of 4M urea into, and assembling needs the regular hour just can obtain stable component, and in assembling process, formed intermediate state (settling ratio 4.5-5.5S).The result also shows, proteic assembling speed of p239 and temperature and (NH ₄) ₂SO ₄Concentration all be proportionate (Fig. 5 A-5F).(1) (NH ₄) ₂SO ₄Concentration is high more, and assembling speed is fast more.Especially, with 0.3M or 0.5M (NH ₄) ₂SO ₄Under the situation about handling, in assembling process, can be observed tangible intermediate state component, and at 1M (NH ₄) ₂SO ₄In institute's inductive assembling process, then do not observe the intermediate state component, this further confirms assembling speed and (NH ₄) ₂SO ₄Concentration is proportionate.(2) temperature is high more, and assembling speed is fast more.Especially, with 0.3M (NH ₄) ₂SO ₄Under the situation about handling, be contained in 25 ℃ of following groups of grains and reach stable after hatching 6 hours, and only needed 4 hours can reach stable 37 ℃ of following particles assemblings.Particle assembling reaches that to stablize the required time following: 0.3M (NH ₄) ₂SO ₄/ 25 ℃ of (6 hours)=0.5M (NH ₄) ₂SO ₄/ 25 ℃ of (6 hours)=1.0M (NH ₄) ₂SO ₄/ 25 ℃ of (6 hours)＞0.3M (NH ₄) ₂SO ₄/ 37 ℃ of (4 hours)＞0.5M (NH ₄) ₂SO ₄/ 37 ℃ of (2 hours)＞1.0M (NH ₄) ₂SO ₄/ 37 ℃ (1 hour).This shows, temperature to the influence of assembling speed greater than (NH ₄) ₂SO ₄The influence of concentration.Reference protein E2 is containing 0.0M, 0.3M, 0.5M or 1.0M (NH ₄) ₂SO ₄4M urea in all do not form grain fraction (Fig. 5 G) in 37 ℃ after hatching 24 hours.Find (NH in addition ₄) ₂SO ₄Concentration is high more, and the proteic settling ratio of p239 particle and E2 is more little, and this shows (NH ₄) ₂SO ₄Measurement to settling ratio has certain interference.

Further the result to Fig. 5 carries out data analysis.Analytical results is shown among Fig. 6.Especially, Fig. 6 A has compared different incubation temperature (25 ℃/37 ℃) and different (NH with Fig. 6 B ₄) ₂SO ₄Under the concentration, the component in the p239 assembling process distributes.As time goes on or the increase of temperature or (NH this result shows, ₄) ₂SO ₄The raising of concentration, the proteic assembling speed of p239 is accelerated, and intermediate state also disappears thereupon, and the proteic assembling process of this explanation p239 is by (NH ₄) ₂SO ₄The endoenergetic process of inductive hydrophobic interaction.Fig. 6 C has shown at 25 ℃ of incubation temperature and different (NH ₄) ₂SO ₄Under the concentration, the change curve of each component in the p239 assembling process.This result shows, at 0.3M and 1.0M (NH ₄) ₂SO ₄Under the concentration, change of component preceding 5 hours in the assembling process is comparatively remarkable and violent, and at 0.5M (NH ₄) ₂SO ₄Under the concentration, assembling process comparatively relaxes, and the variation of various components is more consistent, possibly be more excellent groups of grains process of assembling.Fig. 6 D shown 37 ℃ with different (NH ₄) ₂SO ₄After hatching 24 hours under the concentration, the distribution situation of p239 particle and the proteic settling ratio of E2.This result shows, (NH ₄) ₂SO ₄Concentration is consistent to the trend that influences of p239 particle and the proteic settling ratio of E2: along with (NH ₄) ₂SO ₄The raising of concentration, settling ratio descends; And, because E2 albumen is at different (NH ₄) ₂SO ₄Be dimeric forms under the concentration, therefore, this result also explains at each (NH ₄) ₂SO ₄The p239 particle that obtains under the concentration also is consistent.

The final assembled state of embodiment 3:p239 particulate

The p239 assembling process of describing according to embodiment 2, with p239 albumen at 4M urea+0.5M (NH ₄) ₂SO ₄In under 37 ℃, hatched 12 hours, dialyse then to PBS solution, thereby obtain the final assembled state of p239 particulate.Use methods such as transmission electron microscope observing, subsidence rate analysis, sieve chromatography analysis, dynamic light scattering analysis that p239 particulate graininess is studied.

Transmission electron microscope observing

The 200kV transmission electron microscope that the instrument that uses is produced as company of NEC, magnification is 25,000 times.The p239 particle that obtains with 2% phospho-wolframic acid pH7.0 negative staining, is fixed on the copper mesh of spray charcoal, observes.Electronic Speculum result sees Fig. 7 A, and wherein visible sample major part presents that diameter is about 20nm, virus-like particle of uniform size.

Subsidence rate is analyzed

The instrument that uses is U.S. Beckman XL-A analysis mode ultracentrifuge, and it is furnished with optical detection system and An-50T i and An-60T i rotary head.Described in embodiment 1, adopt sedimentation rate method to analyze p239 particulate settling ratio.The result is shown in Fig. 7 B, and p239 particulate settling ratio is 22.6s.

Sieve chromatography is analyzed

Adopt the 1120 Compact LC performance liquid chromatography chromatographic systems and the TSK Gel PW5000xl 7.8x300mm pillar of German Agilent to carry out the sieve chromatography analysis.Damping fluid 1xPBS pre-balance chromatography column with 2 times of column volumes does not have considerable change until the absorption value at 280nm place.The absorption value of detector is made zero, then by the automatic sampler sample introduction and analyze.The result is shown in Fig. 7 C, and p239 particulate RT is 13.7min.

Dynamic light scattering is measured

The DynaPro MS/X type dynamic light scattering (containing temperature regulator) that the instrument that uses is produced as U.S. Protein Solutions company, the algorithm that uses is the Regulation algorithm.The p239 particulate samples that is obtained is measured behind 0.22 μ m membrane filtration.Measuring result is seen Fig. 7 D.The result shows that the p239 particle is single for component in solution, aquation molecular dynamics radius is the particle of 13.5nm.

The research of the proteic particle composition mechanism of embodiment 4:p239

Circular dichroism spectrum (CD) is analyzed

According to before crystalline structure (.Proc Natl Acad Sci USA (2009) 106:12986-12991 such as Yamashita T of the HEV virus-like particle described; .ProcNatl Acad Sci USA (2009) 106:12992-12997 such as Guu TS) (referring to Fig. 8); P239 albumen is when further assembling becomes particle by dimer; Alpha-helix interacts (Fig. 8 A) on three times of direction of principal axis of icosahedron symplex structure, and the benzene ring side chain of Tyr residue has stabilization (Fig. 8 B) to the orientation of alpha-helix on the beta sheet.Relatively the circular dichroism spectrum (referring to Fig. 3 A and 3B) before and after the assembling of p239 albumen is found: in extreme ultraviolet CD spectrum; Unassembled p239 albumen and bigger through near p239 particle amplitude difference 190nm of assembling, this possibly be because due to assembling occurs near the alpha-helix; In near ultraviolet CD spectrum, unassembled p239 albumen is with less through the p239 particle difference of assembling, and main difference occurs in (seeing Fig. 3 B) near the 275nm, and this possibly change relevant with the hydrophobic environment of Tyr.

The sudden change of the hydrophobic amino acid in the HEV ORF2aa368-aa395 zone is to p239 albumen The influence of particle assembling

P239 albumen and E2 albumen show that in the significant difference aspect the particle assembling HEV ORF2aa368-aa395 is very important for the assembling of HEV VLP.For this reason, the contriver has further analyzed the spontaneous mutation conservative property (referring to table 2) of the amino-acid residue (that is the proteic N terminal sequence of p239) in HEV ORF2 aa368-aa395 zone.The result shows, a plurality of hydrophobic amino acid residues in this zone (Ile or Leu) (in table 2, representing with runic) high conservative.

The natural mutation rate of table 2.p239 albumen n end structural domain (HEV ORF2 aa368-395) amino-acid residue

Further discover the 372nd, 375 and 395 leucine residue (Leu of HEV capsid protein ³⁷², Leu ³⁷⁵And Leu ³⁹⁵) (structure of p239 albumen n end structural domain is as shown in Figure 9, and this structure can be referring to Yamashita etc., Proc Natl Acad Sci U S A, 2009,106:12986-12991 to have vital role for the hydrophobic region of keeping p239 albumen n end structural domain.

Use point mutation process known in the art, with the Leu in the p239 albumen n end structural domain ³⁷², Leu ³⁷⁵And Leu ³⁹⁵Sport hydrophilic amino-acid residue Glu (to destroy its hydrophobicity) respectively, obtain proteic 3 the point mutation albumen of p239, L372E, L375E and L395E.These 3 point mutation albumen are carried out SDS-PAGE analyze, and use viral hepatitis type E reconvalescent's serum (being so kind as to give) to detect their immunoreactivity through the Western engram analysis by the blood station, Xiamen.The result shows that opposite with p239 albumen, these 3 point mutation albumen all can not form dimer, and has lost the reactivity (referring to Figure 10 A) with viral hepatitis type E reconvalescent serum.Further, detect the proteic particle assembling of these 3 point mutation ability through the sieve chromatography analysis.The result shows that opposite with p239 albumen, these 3 point mutation albumen all can not be assembled becomes particle (referring to Figure 10 B).

Above-mentioned result shows that p239 albumen mainly is assembled into VLP through hydrophobic interaction, and hydrophobic interaction is very important for the proteic particle assembling of p239.Further; Result in conjunction with

embodiment

2 and 3 can know; Salt ion (ammonium sulfate for example; Ammonium chloride etc.) interpolation can improve the proteic hydrophobic interaction of p239, promotes the proteic particle assembling of p239, thereby makes that p239 albumen can assembling assembly virus-like particle under the situation that denaturing agent (for example urea) exists.

The present invention has illustrated the composition mechanism of HEV VLP through above-mentioned experiment, and provides new HEV capsid protein or its variant or slice groups are dressed up the method for VLP, and this method can be applicable to the preparation process of hepatitis E vaccine, promotes large-scale industrial production.

Although embodiment of the present invention has obtained detailed description, those skilled in the art will appreciate that according to disclosed all instructions, can carry out various modifications and changes to details, and these change all within protection scope of the present invention.Four corner of the present invention is provided by accompanying claims and any equivalent thereof.

Claims

1. the method for preparing the virus-like particle (VLP) of hepatitis E virus (HEV); It comprises step: under the condition that denaturing agent exists; Make HEV capsid protein or its variant or slice groups dress up VLP through adding salt; Wherein said HEV capsid protein or its variant or fragment have the ability that is assembled into VLP

Said denaturing agent for example can be urea, urea, Guanidinium hydrochloride or its combination, preferred urea;

Said salt for example can be NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, preferred (NH ₄) ₂SO ₄Preferably, the ionic strength of said salt is 0.9mol/kg at least, for example (the NH of 0.3M at least ₄) ₂SO ₄

Said HEV capsid protein or its variant or fragment for example can be with HEV capsid protein or its variant or the fragment of inclusion body formal representation in intestinal bacteria;

The variant of said HEV capsid protein or fragment for example can be p239 albumen, and preferably, the proteic aminoacid sequence of said p239 is shown in SEQ ID NO:2.

2. prepare the method for the virus-like particle (VLP) of hepatitis E virus (HEV), it comprises the steps:

Wherein,

In step 1), for example through with the inclusion body formal representation said HEV capsid protein or its variant or fragment being provided in intestinal bacteria with it;

For example, the variant of said HEV capsid protein or fragment can be p239 albumen, and preferably, the proteic aminoacid sequence of said p239 is shown in SEQ ID NO:2;

In step 2) in, said denaturing agent for example can be urea, urea, Guanidinium hydrochloride or its combination, preferred urea;

In step 3), said salt for example can be NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, preferred (NH ₄) ₂SO ₄Preferably, the ionic strength of said salt is 0.9mol/kg at least, for example (the NH of 0.3M at least ₄) ₂SO ₄

3. control HEV capsid protein or its variant or slice groups are dressed up the method for the assembling speed of VLP; It comprises step: control comprises the salt concn of HEV capsid protein or its variant or segmental solution and/or controls the temperature of said solution; Wherein said HEV capsid protein or its variant or fragment have the ability that is assembled into VLP

The variant of said HEV capsid protein or fragment for example can be p239 albumen, and preferably, the proteic aminoacid sequence of said p239 is shown in SEQ ID NO:2;

Said solution comprises or does not comprise denaturing agent;

Said salt for example can be NaCl, NH ₄Cl, (NH ₄) ₂SO ₄, Na ₂SO ₄Or its combination, preferred (NH ₄) ₂SO ₄

Wherein, when the salt concn of said solution raises and/or the temperature of said solution when raising, said assembling speed is accelerated.

4. salt is used to promote to have HEV capsid protein or its variant of the ability that is assembled into VLP or the purposes that slice groups is dressed up VLP, wherein,

Said salt can be used to promote the assembling of VLP under denaturing agent existence or non-existent condition;

Said denaturing agent is urea, urea, Guanidinium hydrochloride or its combination for example, preferred urea.

5. destroy the method for HEV capsid protein or its variant or segmental particle assembling ability, it comprises the hydrophobic amino acid that is arranged in the zone corresponding with HEV ORF2 aa368-395 in HEV capsid protein or its variant or the fragment is sported hydrophilic amino acid,

For example, the variant of said HEV capsid protein or fragment can be p239 albumen; Preferably, the proteic aminoacid sequence of said p239 is shown in SEQ ID NO:2;

For example, the said hydrophobic amino acid that is arranged in the zone corresponding the 372nd, 375 or 395 leucine that can be HEV ORF2 with HEV ORF2 aa368-395; For example, said leucine can be sported L-glutamic acid.

6. promote HEV capsid protein or its variant or slice groups to dress up the method for VLP, it comprises the hydrophilic amino acid that is arranged in the zone corresponding with HEV ORF2 aa368-395 in HEV capsid protein or its variant or the fragment is sported hydrophobic amino acid.

7. preparation is used to prevent and/or treat the method for the vaccine of hepatitis E, and it comprises:

1) method according to claim 1 or 2 prepares the HEV virus-like particle;